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Mutations in the human Zip4 gene cause acrodermatitis enteropathica, a rare, pseudo-dominant, lethal genetic disorder. We created a tamoxifen-inducible, enterocyte-specific knockout of this gene in mice which mimics this human disorder. We found that the enterocyte Zip4 gene in mice is essential throughout life, and loss-of-function of this gene rapidly leads to wasting and death unless mice are nursed or provided excess dietary zinc. An initial effect of the knockout was the reprogramming of Paneth cells, which contribute to the intestinal stem cell niche in the crypts. Labile zinc in Paneth cells was lost, followed by diminished Sox9 (sex determining region Y-box 9) and lysozyme expression, and accumulation of mucin, which is normally found in goblet cells. This was accompanied by dysplasia of the intestinal crypts and significantly diminished small intestine cell division, and attenuated mTOR1 activity in villus enterocytes, indicative of increased catabolic metabolism, and diminished protein synthesis. This was followed by disorganization of the absorptive epithelium. Elemental analyses of small intestine, liver, and pancreas from Zip4-intestine knockout mice revealed that total zinc was dramatically and rapidly decreased in these organs whereas iron, manganese, and copper slowly accumulated to high levels in the liver as the disease progressed. These studies strongly suggest that wasting and lethality in acrodermatitis enteropathica patients reflects the loss-of-function of the intestine zinc transporter ZIP4, which leads to abnormal Paneth cell gene expression, disruption of the intestinal stem cell niche, and diminished function of the intestinal mucosa. These changes, in turn, cause a switch from anabolic to catabolic metabolism and altered homeostasis of several essential metals, which, if untreated by excess dietary zinc, leads to dramatic weight loss and death.

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates. In the last few decades, technical advances in altering the genes of organisms have led to many discoveries about how genes work. For example, it is now possible to add a specific DNA sequence to a gene so that the protein it makes will carry a ‘tag’ that enables us to track it in cells. One such tag is called green fluorescent protein (GFP) and it is often used to study other proteins in living cells because it produces green fluorescence that can be detected under a microscope. It is labor intensive to add tags to individual genes, so this limits the number of proteins that can be studied in this way. In 2011, researchers developed a new method that can easily tag many genes in fruit flies. It makes use of small sections of DNA called transposons, which are able to move around the genome by ‘cutting’ themselves out of one location and ‘pasting’ themselves in somewhere else. The researchers used a transposon called Minos, which is naturally found in fruit flies. When Minos inserts into a gene, it often disrupts the gene and stops it from working. However, the researchers could swap the inserted transposon for a gene encoding GFP by making use of a natural process that rearranges DNA in cells. This resulted in the protein encoded by the gene containing GFP and so it can be detected under a microscope. This method allowed the researchers to create a collection of fly lines that have the GFP tag on many different proteins. Now, Nagarkar-Jaiswal et al. have greatly expanded this initial collection. More than 75% of GFP-tagged proteins worked normally and the flies producing these altered proteins remain healthy. It is possible to use a technique called RNA interference against the GFP to lower the production of the tagged proteins. Moreover, Nagarkar-Jaiswal et al. show that it is also possible to degrade the tagged proteins so that less protein is present. The removal of proteins is reversible and can be done in specific tissues during any phase in fly development. These techniques allow researchers to directly associate the loss of the protein with the consequences for the fly. This collection of fruit fly lines is a useful resource that can help us understand how genes work. The method for tagging the proteins could also be modified to work in other animals.

We describe a transgenesis platform for Drosophila melanogaster that integrates three recently developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), recombineering, and bacteriophage ΦC31-mediated transgenesis. The BAC is maintained at low copy number, facilitating plasmid maintenance and recombineering, but is induced to high copy number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap repair efficiently retrieves DNA fragments up to 133 kilobases long from P1 or BAC clones. ΦC31-mediated transgenesis integrates these large DNA fragments at specific sites in the genome, allowing the rescue of lethal mutations in the corresponding genes. This transgenesis platform should greatly facilitate structure/function analyses of most Drosophila genes.

In vitro derivation of pancreatic β-cells from human pluripotent stem cells holds promise as diabetes treatment. Despite recent progress, efforts to generate physiologically competent β-cells are still hindered by incomplete understanding of the microenvironment’s role in β-cell development and maturation. Here, we analyze the human mesenchymal and endothelial primary cells from weeks 9-20 fetal pancreas and identify a time point-specific microenvironment that permits β-cell differentiation. Further, we uncover unique factors that guide in vitro development of endocrine progenitors, with WNT5A markedly improving human β-cell differentiation. WNT5A initially acts through the non-canonical (JNK/c-JUN) WNT signaling and cooperates with Gremlin1 to inhibit the BMP pathway during β-cell maturation. Interestingly, we also identify the endothelial-derived Endocan as a SST+ cell promoting factor. Overall, our study shows that the pancreatic microenvironment-derived factors can mimic in vivo conditions in an in vitro system to generate bona fide β-cells for translational applications. In vitro differentiation of pancreatic beta cells offers a potential therapeutic approach for diabetes. Here they show human pluripotent stem cell derived pancreatic progenitors differentiate into insulin-secreting cells by crosstalk of WNT5A and BMP signaling.

Described is a randomly inserting transposon that can be swapped for gene traps, coding insertions or protein tag genes, thus expanding the toolkit for Drosophila melanogaster genome engineering. We demonstrate the versatility of a collection of insertions of the transposon Minos -mediated integration cassette (MiMIC), in Drosophila melanogaster . MiMIC contains a gene-trap cassette and the yellow + marker flanked by two inverted bacteriophage ΦC31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNA manipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaster toolkit.

Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multiplexing in post-lysis endpoint luciferase assays from two to six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed solotransfection, minimizing experimental errors. We engineer a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We can monitor effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, and its broad application across different research fields. Multiplexed detection of luciferase-based sensors in the same sample is challenging and limited by the substrates’ emission spectra. Here the authors establish a system based on three different luciferases and sequential detection to achieve measurements of up to six parameters within the same experiment.

Two bacterial artificial chromosome (BAC) libraries, spanning almost the entire D. melanogaster genome in insert sizes of 20 and 80 kb, that allow easy integration into the fruit fly genome at defined docking sites provide a rich resource to study gene expression and function. We constructed Drosophila melanogaster bacterial artificial chromosome libraries with 21-kilobase and 83-kilobase inserts in the P[acman] system. We mapped clones representing 12-fold coverage and encompassing more than 95% of annotated genes onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using ΦC31 integrase to rescue mutations. They can be modified through recombineering, for example, to incorporate protein tags and assess expression patterns.

To achieve the ultimate goal of personalized treatment of patients, accurate molecular diagnosis and precise interpretation of the impact of genetic variants on gene function is essential. With sequencing cost becoming increasingly affordable, the accurate distinguishing of benign from pathogenic variants becomes the major bottleneck. Although large normal population sequence databases have become a key resource in filtering benign variants, they are not effective at filtering extremely rare variants. To address this challenge, we developed a novel statistical test by combining sequencing data from a patient cohort with a normal control population database. By comparing the expected and observed allele frequency in the patient cohort, variants that are likely benign can be identified. The performance of this new method is evaluated on both simulated and real data sets coupled with experimental validation. As a result, we demonstrate this new test is well powered to identify benign variants, and is particularly effective for variants with low frequency in the normal population. Overall, as a general test that can be applied to any type of variants in the context of all Mendelian diseases, our work provides a general framework for filtering benign variants with very low population allele frequency.

The Drosophila Ten-m (also called Tenascin-major, or odd Oz (odz)) gene has been associated with a pair-rule phenotype. We identified and characterized new alleles of Drosophila Ten-m to establish that this gene is not responsible for segmentation defects but rather causes defects in motor neuron axon routing. In Ten-m mutants the inter-segmental nerve (ISN) often crosses segment boundaries and fasciculates with the ISN in the adjacent segment. Ten-m is expressed in the central nervous system and epidermal stripes during the stages when the growth cones of the neurons that form the ISN navigate to their targets. Over-expression of Ten-m in epidermal cells also leads to ISN misrouting. We also found that Filamin, an actin binding protein, physically interacts with the Ten-m protein. Mutations in cheerio, which encodes Filamin, cause defects in motor neuron axon routing like those of Ten-m. During embryonic development, the expression of Filamin and Ten-m partially overlap in ectodermal cells. These results suggest that Ten-m and Filamin in epidermal cells might together influence growth cone progression.

Key Points Many genetic tools have been developed over the past 100 years to manipulate the genome of the fruitfly Drosophila melanogaster ; despite the success of most of these methods, some limitations need to be overcome. The past 3 years have seen the introduction of several new technologies that allow flies to be manipulated more easily than any other multicellular organism. The new methods include the ability to create molecularly designed deletions, improved genetic-mapping technologies, strategies for creating targeted mutations, new transgenic approaches and the ability to manipulate large fragments of DNA. Two transposable elements, P -elements and piggyBac s, each having different properties and mobilization characteristics, are being used to generate insertions in every gene of the D. melanogaster genome. Two P -elements, located at different positions in cis or in trans on the same homologous pair of chromosomes, can be used to generate transposon-induced deficiencies. A P -element that contains an internal hobo transposable element allows nested deletions to be generated from a common starting point ('deletion-generator' technology). Two transposons, each of which contains an FRT site, are used to create molecularly defined deletions through Flp-mediated excision of the intervening sequence. SNPs and molecularly defined P -element insertions improve and accelerate gene-mapping efforts. 'Ends-out replacement gene targeting' removes a desired genomic sequence through homologous recombination. Target-induced local lesions in genomes (TILLING), a technique that was first applied in Arabidopsis thaliana , quickly and efficiently identifies single-nucleotide changes in specific genes and might allow the isolation of an allelic series for structure–function analysis. The bacteriophage φC31 efficiently integrates DNA at defined loci in the fly genome. Recombineering facilitates gap-repair-mediated subcloning and the subsequent mutagenesis of large DNA fragments. The popularity of Drosophila melanogaster as a model for understanding eukaryotic biology over the past 100 years has been accompanied by the development of numerous tools for manipulating the fruitfly genome. Here we review some recent technologies that will allow Drosophila melanogaster to be manipulated more easily than any other multicellular organism. These developments include the ability to create molecularly designed deletions, improved genetic mapping technologies, strategies for creating targeted mutations, new transgenic approaches and the means to clone and modify large fragments of DNA.